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Journal of Molecular S Iyer and S K Agarwal Epigenetic regulation in 61:1 R13–R24 Endocrinology endocrine cells REVIEW Epigenetic regulation in the tumorigenesis of MEN1-associated endocrine cell types

Sucharitha Iyer and Sunita K Agarwal

Metabolic Diseases Branch, National Institute of Diabetes and Digestive and Kidney Diseases, NIH, Bethesda, Maryland, USA

Correspondence should be addressed to S K Agarwal: [email protected]

Abstract

Epigenetic regulation is emerging as a key feature in the molecular characteristics of Key Words various human diseases. Epigenetic aberrations can occur from mutations in genes ff neuroendocrine associated with epigenetic regulation, improper deposition, removal or reading of ff pancreas histone modifications, DNA methylation/demethylation and impaired non-coding ff parathyroid RNA interactions in chromatin. Menin, the protein product of the gene causative for ff pituitary the multiple endocrine neoplasia type 1 (MEN1) syndrome, interacts with chromatin- associated protein complexes and also regulates some non-coding RNAs, thus participating in epigenetic control mechanisms. Germline inactivating mutations in the MEN1 gene that encodes menin predispose patients to develop endocrine tumors of the parathyroids, anterior pituitary and the duodenopancreatic neuroendocrine tissues. Therefore, functional loss of menin in the various MEN1-associated endocrine cell types can result in epigenetic changes that promote tumorigenesis. Because epigenetic changes are reversible, they can be targeted to develop therapeutics for restoring the tumor epigenome to the normal state. Irrespective of whether epigenetic alterations are the cause or consequence of the tumorigenesis process, targeting the endocrine tumor- associated epigenome offers opportunities for exploring therapeutic options. This review presents epigenetic control mechanisms relevant to the interactions and targets of menin, and the contribution of epigenetics in the tumorigenesis of endocrine cell types Journal of Molecular from menin loss. Endocrinology (2018) 61, R13–R24

Introduction

Epigenetic changes observed in various human diseases regulation in tumor etiology is highlighted by the have been shown to play important roles as disease discovery of mutations in genes encoding factors that drivers, biomarkers and therapeutic targets. Epigenetic participate in epigenetic regulatory mechanisms in changes can occur due to mutations in factors that various cancers (Koschmann et al. 2017). Menin, the control epigenetic modifications or in response to protein encoded by the gene responsible for causing the metabolic or environmental signals, which lead to multiple endocrine neoplasia type 1 (MEN1) syndrome, improper deposition, removal or reading of histone participates in epigenetic regulation (Shilatifard 2012, van modifications, DNA methylation/demethylation and Nuland et al. 2013). Patients with the MEN1 syndrome impaired non-coding RNA interactions in chromatin carry germline heterozygous inactivating mutations (Allis et al. 2015). The evidence for the role of epigenetic in the MEN1 gene located at chromosome 11q13, that

-18-0050 Journal of Molecular S Iyer and S K Agarwal Epigenetic regulation in 61:1 R14 Endocrinology endocrine cells encodes menin, which predisposes to the development of could potentially promote tumorigenesis. Furthermore, tumors in multiple endocrine organs (Chandrasekharappa evidence from genome and exome sequencing efforts et al. 1997, Lemmens et al. 1997). The main MEN1- of sporadic non-functioning and functioning PNETs, associated endocrine cell types that form tumors are parathyroid adenoma and lung neuroendocrine tumors located in the parathyroids, anterior pituitary and the have shown additional somatic mutations in other genes duodenopancreatic neuroendocrine tissues (Thakker associated with epigenetic control mechanisms that et al. 2012). Other MEN1-associated endocrine tumors predict epigenetic changes to promote tumorigenesis in include foregut carcinoids that arise from neuroendocrine these endocrine cell types (Jiao et al. 2011, Cromer et al. cells of the lung, thymus or gastrointestinal (GI) tract. 2012, Chou et al. 2016, Romano et al. 2017, Scarpa et al. The tumors are classified as functioning when they 2017, Simbolo et al. 2017, Wang et al. 2017). hypersecrete the hormones characteristic of the endocrine Menin is a 67 kDa ubiquitously expressed cell types affected, or non-functioning when they do predominantly nuclear protein, with no intrinsic not hypersecrete any hormones. Parathyroid tumors enzymatic activity. As stated earlier, menin is a tumor are functioning causing primary hyperparathyroidism suppressor in the context of MEN1-associated endocrine due to excessive parathyroid hormone (PTH) secretion. cell types; however, menin has been shown to act as a pro- Pituitary and duodenopancreatic neuroendocrine tumors oncogenic co-factor in mixed lineage leukemia (Yokoyama in MEN1 patients are observed to be either functioning et al. 2005, Muntean & Hess 2012). The crystal structure (prolactinoma, 20%; somatotropinoma, 10%; gastrinoma, of menin shows a deep pocket that can act as a binding 40%; insulinoma, 10%) or non-functioning (pituitary, site for interacting proteins (Protein Data Bank No. 3RE2 5%; PNETs, 20–55%) (Schussheim et al. 2001, Thakker and 3U84) (Murai et al. 2011, Huang et al. 2012). Evidence et al. 2012, Marini et al. 2017). In the context of these from protein–protein interaction studies have shown endocrine tumors, MEN1 acts as a classic tumor suppressor that menin is involved in epigenetic regulation and gene because human MEN1-associated tumors show loss gene transcription through interaction with proteins in of heterozygosity (LOH) at 11q13 resulting in biallelic chromatin-associated protein complexes and transcription loss/inactivation of MEN1 (Chandrasekharappa et al. factors, affecting the expression of target genes such as 1997, Lemmens et al. 1997). Similarly, in genetically genes that control cell proliferation (Karnik et al. 2005, Wu engineered mouse models, germline targeted deletion of & Hua 2011). Interacting partners of menin also include both copies of the Men1 gene causes embryonic lethality, proteins that participate in cell adhesion, cell division, while germline targeted deletion of one copy of the cell motility, cell signaling, cytoskeletal structure, DNA Men1 gene generates live mice that later in life develop repair in response to DNA damage and studies conducted endocrine tumors similar to those seen in the human in Drosophila melanogaster have shown a role of menin MEN1 syndrome with LOH at the Men1 locus (Agarwal in the maintenance of genomic stability (Busygina et al. 2014, Agarwal 2017b). 2004, Hendy et al. 2009, Agarwal 2017a, Feng et al. 2017a). Interestingly, somatic MEN1 mutations are also Investigations to elucidate the biological function of detected in 30–40% of sporadic tumors of MEN1- menin through protein–protein interaction studies have associated endocrine cell types – parathyroid adenomas, significantly advanced our knowledge about the potential gastrinomas, pancreatic neuroendocrine tumors (PNETs) roles of menin in various cellular pathways, but they have and lung neuroendocrine tumors (Zhuang et al. 1997, been mainly conducted using cell lines (HEK293, HeLa, Debelenko et al. 1997, Jiao et al. 2011, Cromer et al. 2012, HepG2, K562 and mouse embryo fibroblasts (MEFs)) that Newey et al. 2012, Scarpa et al. 2017, Simbolo et al. 2017). are unrelated to MEN1-associated endocrine cell types. Among sporadic PNETs, MEN1 mutations are detected in Follow-up functional characterization of some interactions the non-functioning PNETs, but rarely in the functioning of menin have been performed in endocrine/endocrine- PNETs (insulinomas) (Jiao et al. 2011, Cao et al. 2013, related cell lines (βlox5, BON1, INS-1, MIN6, NIH-H720, Lichtenauer et al. 2015, Chou et al. 2016, Scarpa et al. NIH-727, and QGP1) albeit not in endocrine cell lines 2017, Wang et al. 2017). Also, MEN1 mutations are rarely with MEN1 mutations or that lack menin because no such detected in sporadic pituitary tumors (Poncin et al. 1999, cell lines are currently available. The focus of this review Newey et al. 2013, Bi et al. 2017, Marques & Korbonits will be on studies conducted using endocrine/endocrine- 2017). Because menin participates in epigenetic regulation, related cell lines or menin-null endocrine tumors from functional loss of menin in the various MEN1-associated mouse models. This review excludes the discussion of endocrine cell types would lead to epigenetic changes that menin’s interaction with transcription factors because

Eukaryotic DNA with core histone proteins (H2A, H2B, H3 and H4) is packaged into nucleosomes to form chromatin fibers (Olins & Olins 2003). This packaging regulates access to DNA for various biological processes such as DNA replication, DNA repair and transcription into RNA. Modifications to chromatin are essential to permit or restrict access, for example, to the transcriptional machinery including transcription factors for gene expression (Flavahan et al. 2017). Chromatin modifiers and remodelers constitute ‘epigenetic factors’ that function in multi-protein complexes that may also include non- coding RNAs, to modify the DNA or histones contained in nucleosomes (Allis & Jenuwein 2016). The multi- protein complexes contain enzymes and proteins referred to as the ‘writers’, ‘readers’ and ‘erasers’ of chromatin modifications Allis( & Jenuwein 2016). Modifications to DNA include methylation, hydroxymethylation and further oxidation. Histones can undergo a variety of post- translational modifications (PTMs) such as methylation, acetylation, phosphorylation and ubiquitination to generate an active, repressive or poised state to control gene expression; and the array of PTMs that govern a specific chromatin state is referred to as the ‘histone code’ (Huang et al. 2014). Also, chromatin interactions can occur over short and long distances thereby generating conditions that enhance gene transcription or insulate the regions of gene transcription (Rowley & Corces 2016). ‘Epigenetic changes’ through alterations in DNA and histone modifications, chromatin remodeling and alterations in non-coding RNA interactions, create a Figure 1 Overview of menin-dependent epigenetic regulation in MEN1-associated positive or negative environment affecting the outcome endocrine cell types. Epigenetic mechanisms and the observed or of gene transcription and chromatin structure (Allis et al. predicted consequence of menin loss are shown that were investigated in 2015). The ‘epigenetic marks’ on DNA and histones that various studies of human or mouse endocrine tumors or studies conducted in endocrine/endocrine-related cell lines. Green or red are generated as a result of the enzymatic activity of various indicates the normal effect of the epigenetic event resulting in the chromatin-modifying enzymes can survive successive activation or repression of gene expression, respectively. Up or down cell divisions, and they are reversible, which presents arrow indicates increase or decrease, respectively, of the epigenetic event in a cell line or tumor with menin deficiency. ALT (alternative lengthening an opportunity for correction to the normal state, for of telomeres) is shown in blue (and not in red or green) because it is an example, when such changes promote disease processes event associated with chromatin structure at telomeres.

Histone methylation and demethylation Interestingly, both KMT2A and MEN1 co-localize at human chromosome 11q and mouse chromosome 19. LOH at Methylation of histones at lysine or arginine residues this locus in MEN1 mutation-positive human PNETs and in the chain of amino acids that protrude from the mouse PNETs (insulinomas) in Men1+/− mice contributes to nucleosome, referred to as the ‘histone tail’, is achieved the tumorigenesis process. Targeted conditional deletion by histone methyltransferases (KMTs) that exist in of both Kmt2a and Men1 in mice (Kmt2af/f;Men1f/f;RIP-Cre) multi-subunit protein complexes (Cheng 2014). KMTs led to the acceleration of pancreatic islet tumor formation methylate histone H3 on lysine residues at positions 4, 9, and also shortened the average life span of these mice (Lin 27, 36 and 79 in the in mono-, di- or tri-methylated form et al. 2016). This study showed that MLL1 can cooperate depending on whether one, two or three methyl groups with menin to prevent tumor formation. In another study, are added, respectively (Chi et al. 2010). In general, the targeted conditional deletion in mice of Men1 and Rbp2, histone H3 lysine-4 trimethyl mark (H3K4me3) specifies which encodes a H3K4me3 demethylase (Rbp2f/f;Men1f/ a state of transcriptional activation, which is located at f;RIP-Cre) decreased the rate of tumor formation and the transcriptional start site (TSS) of actively transcribed prolonged survival (Lin et al. 2011). These data showed genes. On the other hand, H3K9me3 and H3K27me3 that the histone demethylase activity of RBP2 is linked to are associated with transcriptional silencing. When both islet tumorigenesis downstream of menin loss. H3K4me3 and H3K27me3 occur together, the gene is said To investigate the contribution of menin-MLL1- to be bivalently poised – a state maintained, for example, mediated epigenetic regulation in tumor formation, by embryonic stem cells during lineage differentiation and genome-wide distribution of the gene activation mark development, to enable rapid switching between active H3K4me3 and its counterpart recessive mark H3K27me3 and repressive gene transcription states (Voigt et al. 2013). was examined in menin-deficient pancreatic islets of Eraser KDMs such as lysine-specific demethylase 1 (LSD1/ 2-month-old Men1f/f;RIP-Cre and control wild-type KDM1A), lysine-specific demethylase 2 (LSD2/KDM1B) mice (Lin et al. 2015). This study found that within and Jumonji AT-rich interacting domain 1A (JARID1A/ the same set of genes, loss of H3K4me3 correlated with KDM5A/RBP2), remove mono- , di- or tri-methylation of increased H3K27me3 in menin-deficient islets, and the H3K4 or H3K9 (Cheng 2014). Depending on the specific expression of such genes was downregulated. The most amino acid residue methylated or demethylated, and the significantly downregulated gene in menin-deficient level of methylation, the process of histone methylation islets was Igfbp2, which also displayed loss of H3K4me3 and demethylation generates active or repressive together with gain of H3K27me3. Interestingly, the chromatin states. decline in Igfbp2 expression in menin-deficient islets could be reversed with simultaneous deletion of Rbp2 (Lin et al. 2015). This observation further confirms that H3K4me3 and H3K27me3 the histone demethylase activity of RBP2 is linked to islet There are at least six different protein complexes tumorigenesis downstream of menin loss. that contain KMTs for catalyzing the gene-activating Genome-wide occupancy patterns of menin, MLL1 histone modification H3K4me3 Dreijerink( et al. 2017). and another component of the MLL-protein-complex Menin has been shown to interact with KMTs, MLL1 (RBBP5) in human pancreatic islet cells, combined with (KMT2A) and MLL2 (KMT2B), in two of those multi- gene expression analysis in menin-null islets from Men1f/ subunit protein complexes, and studies using chromatin f;RIP-Cre mice showed upregulation of the pancreatic immunoprecipitation have shown that menin is present islet β-cell differentiation factor HLXB9 (Scacheri et al. at the TSS with H3K4me3 (Hughes et al. 2004, Yokoyama 2006). Candidate gene analysis using menin-null islets et al. 2004, Scacheri et al. 2006). Several lines of evidence from Men1f/f;RIP-Cre mice showed downregulation of have established the importance of this interaction of cell cycle inhibitory genes in menin-null islet tumors menin involving H3K4me3 and the target genes affected compared to normal islets, mainly the cyclin-dependent using MEN1-associated endocrine cell types, mainly kinase inhibitor (CDKI) genes CDKN2C/p18 and pancreatic islets. CDKN1B/p27, with concomitant loss of H3K4me3 in Mice with targeted deletion of Men1 in the germline their promoter regions (Karnik et al. 2005). In another (Men1+/−) or specifically in the pancreatic isletβ -cells study, a long non-coding RNA, MEG3 was identified as an (Men1f/f;RIP-Cre) develop insulin-secreting pancreatic epigenetic target of menin (Agarwal & Jothi 2012). This islet tumors (insulinomas) (Agarwal 2014, 2017b). study used genome-wide assessment of H3K4me3 and

http://jme.endocrinology-journals.org © 2018 Society for Endocrinology https://doi.org/10.1530/JME-18-0050 Published by Bioscientifica Ltd. Printed in Great Britain Downloaded from Bioscientifica.com at 10/03/2021 12:22:59PM via free access Journal of Molecular S Iyer and S K Agarwal Epigenetic regulation in 61:1 R17 Endocrinology endocrine cells gene expression from wild-type mouse embryonic stem and symmetric dimethylation of arginines. In mammalian cells (mESCs) and menin-null mESCs. Loss of H3K4me3 cells, PRMT5 is the predominant type II arginine was observed in menin-null mESCs at the Meg3 gene, methyltransferase, which acts as a transcriptional together with downregulation of the Meg3 transcript. repressor specifying H3R2me2 and H4R3me2 (Stopa et al. Further studies showed decreased MEG3 levels in mouse 2015). Menin has been shown to interact with PRMT5, and human islet tumors (Modali et al. 2015). Also, loss coinciding with a repressive chromatin mark, symmetric of MEG3 expression was reported in sporadic non- dimethylation of H4R3 (H4R3me2s), at the promoter of the functioning pituitary tumors (Zhao et al. 2005). When growth arrest-specific 1 (Gas1) gene (Gurung et al. 2013). mESCs were in vitro differentiated into pancreatic islet like GAS1 has been shown to direct the binding of the sonic endocrine cells (PILECs), reduced H3K4me3 and reduced hedgehog (SHH) ligand to its receptor patched 1 (PTCH1) gene expression was observed of the Hox genes in PILECs to activate the pro-proliferative and oncogenic Hedgehog derived from menin-null mESCs (Agarwal & Jothi 2012). signaling pathway (Martinelli & Fan 2007). Thus, menin HOX genes have been identified as the direct target of the loss would coincide with increased GAS1 protein and MLL1-complex in many different cell types, particularly oncogenic Hedgehog signaling. Further characterization in hematopoetic cells where the menin-MLL association of menin/PRMT5 showed that pharmacologic inhibition partners as an oncogenic complex in MLL-fusion leukemia of Hedgehog signaling with the drug GDC-0449 reduced (Muntean & Hess 2012). It is important to note that the cell proliferation in insulinoma tumors of Men1f/f;RIP- PILECs were heterogeneous in terms of the cell population Cre mice (Gurung et al. 2013). These data show that as assessed by the expression of pancreatic marker genes, inhibition of Hedgehog signaling in PNETs could target and not entirely consisted of pancreatic endocrine cells the downstream effects from loss of menin/PRMT5 (Agarwal & Jothi 2012). Whether HOX genes play a role interaction, revealing a potential target for treating MEN1 in MEN1-associated endocrine tumorigenesis remains to tumors. be determined. Histone acetylation and deacetylation H3K9me3 Histone acetyl transferases (HATs) catalyze acetylation Another histone lysine modification regulated by menin at lysine residues in histone tails. There are five well- is the repressive H3K9me3, through interaction with a characterized HAT families that can acetylate specific lysine methyltransferase, suppressor of variegation 3–9 lysine residues of mammalian histones – p300/CBP, Gcn5- homolog 1 (SUV39H1) (Yang et al. 2013). In INS-1 rat related N-acetyltransferase (GNAT) (GCN5/PCAF), MYST insulinoma cells (Merglen et al. 2004), enhanced H3K9me3 (for MOZ, Ybf2/Sas3, Sas2 and Tip60)-related HATs, the was detected at the promoter region of the membrane general transcription factor HATs, which include the TFIID metalloproteinase (Mme) gene with combined presence of subunit TAF250 and nuclear/steroid hormone receptor menin, SUV39H1 and the chromatin-remodeling protein coactivators (SRCs) (Marmorstein & Zhou 2014). The DAXX (Feng et al. 2017b). The DAXX gene is frequently acetylation marks on histones are biologically read by the mutated in sporadic non-functioning PNETs (Jiao et al. bromodomain (BRD) contained in the bromodomain and 2011, Chou et al. 2016, Scarpa et al. 2017). Therefore, extraterminal (BET) proteins comprising members BRD2, this study helps to integrate the potential involvement of BRD3, BRD4 and BRDT. Histone acetylation generally multiple epigenetic regulators to prevent tumorigenesis. favors active transcription that is reversed by the activity of histone deacetylases (HDACs) (Marmorstein & Zhou 2014). There are 18 HDACs that belong to four different H4R3me2s subtypes classified on sequence similarities, shared PRMTs methylate arginine residues in histone H3 at catalytic mechanisms and active sites (Seto & Yoshida positions 2, 8, 17 and 26; and histones H4 and H2A at 2014). Small-molecule inhibitors (CPI-203, JQ1 and PFI- positions 3 and 11, respectively (Yang & Bedford 2013). 1) of bromodomain interactions with acetylated histone PRMTs are classified into two families. The type I family tails have been developed as potential therapeutics (Perez- comprises the members 1, 2, 3, 4, 6 and 8, which catalyze Salvia & Esteller 2017). JQ1 binds to the acetyl-lysine mono-methylation and asymmetric dimethylation recognition pocket of bromodomains 1 and 2 on BRD4, of arginines (Yang & Bedford 2013). The type II family which displaces BRD4 from chromatin, thus preventing consisting of PRMTs 5, 7 and 9 mediate monomethylation the ‘reading’ of the acetyl mark and blocking active

http://jme.endocrinology-journals.org © 2018 Society for Endocrinology https://doi.org/10.1530/JME-18-0050 Published by Bioscientifica Ltd. Printed in Great Britain Downloaded from Bioscientifica.com at 10/03/2021 12:22:59PM via free access Journal of Molecular S Iyer and S K Agarwal Epigenetic regulation in 61:1 R18 Endocrinology endocrine cells transcription. CPI-203 is a primary amide analog of JQ1, The evidence for the involvement of histone and PFI-1 inhibits BRD2 and BRD4 (Perez-Salvia & Esteller remodeling in MEN1-associated endocrine cells comes 2017). from sporadic non-functioning PNETs that show somatic There is no direct evidence in MEN1-associated mutations in death-domain-associated protein (DAXX) endocrine cells for the interaction of menin in protein and its interacting partner, alpha thalassemia/mental complexes that are involved in specific histone acetylation retardation syndrome X-linked (ATRX) (Jiao et al. 2011, or deacetylation. However, there is indirect evidence for Chou et al. 2016, Scarpa et al. 2017). ATRX with its the involvement of histone acetylation/deacetylation in intrinsic ATPase activity, in collaboration with its partner endocrine cells that comes from the demonstration of DAXX, a highly specific histone chaperone, deposit the growth inhibitory effects of HDAC inhibitors (HDCAi) histone variant H3.3 in chromatin at telomeric and (trichostatin-A (TSA), thailandepsin‐A (TDP‐A), sodium pericentromeric regions (Amorim et al. 2016). Telomeres butyrate (NaB), valproic acid (VPA) and MS-275), and BET protect chromosome ends to prevent the loss of DNA, protein bromodomain inhibitors (BETi) (CPI201, PFI-1 and in neoplastic cells, the maintenance of telomeres is and JQ1) in neuroendocrine tumor cell lines BON1, NCI- required for immortality. Lengthening of telomeres in H720, NCI-H727 and QGP1 (Baradari et al. 2006, Adler tumor cells can occur from excessive production of the et al. 2009, Wong et al. 2014, Arvidsson et al. 2016, Lines telomerase enzyme or through alternative lengthening et al. 2017). BON1 is a human cell line derived from a of telomeres (ALT), a process similar to homologous peripancreatic lymph node metastasis of a pancreatic recombination (Amorim et al. 2016). ATRX/DAXX neuroendocrine carcinoid tumor (Townsend et al. 1993). mutations or loss of their expression in sporadic PNETs NCI-H720 and NCI-H727 are derived from human atypical correlate with increased telomere length that can and typical bronchial carcinoid tumors, respectively potentially support tumor progression (Kim et al. 2017, (ATCC). QGP-1 is a carcinoembryonic antigen-producing Marinoni et al. 2017, Scarpa et al. 2017, Singhi et al. 2017, cell line from a human pancreatic islet carcinoma (Kaku VandenBussche et al. 2017). Some level of ALT was also et al. 1980). Anti-tumor activity of the HDACi VPA and detected in MEN1 mutation-positive sporadic PNETs BETi CPI203 was also shown in a BON1 xenograft model (Scarpa et al. 2017). Whether ALT plays a role in the (Greenblatt et al. 2007, Wong et al. 2014). Also, in vivo anti- endocrine tumors of patients with the MEN1 syndrome tumor activity of JQ1 has been demonstrated in primary remains to be determined. tumors (insulinomas) of the Men1f/f;RIP-Cre mouse model (Lines et al. 2017). These studies highlight the potential of DNA methylation and demethylation targeting the epigenetic histone acetyl marks in treating MEN1-associated neuroendocrine tumors. DNA methylation is an epigenetic modification that occurs predominantly at CpG sites where a cytosine nucleotide is followed by a guanine nucleotide in which Chromatin remodeling the cytosine is methylated to 5-methylcytosine (5-mC) (Li Histones and nucleosomes are subjected to remodeling & Zhang 2014). Methylation at CpG islands (GC-rich DNA by protein complexes that contain ATP-dependent regions) is often located in promoter regions of genes. chromatin-remodeling activity. Also, eukaryotic cells Aberrant hypo- or hyper-methylation of these promoter express histone variants, in addition to the ‘canonical’ region CpG islands can result in gene overexpression core histones, providing another layer of epigenetic or gene silencing, respectively. A family of DNA methyl regulation. Chromatin-remodeling activity facilitates transferases (DNMTs), designated DNMT1, DNMT3A and transcriptional initiation and elongation for the passage DNMT3B, mediates CpG methylation. While DNMT3A of RNA polymerase through nucleosomal arrays (Clapier and DNMT3B establish de novo DNA methylation, & Cairns 2009). This is achieved by displacing histone DNMT1 serves to propagate and stably maintain the DNA dimers H2A/H2B or H3/H4 leading to the substitution of methylation mark (Li & Zhang 2014). DNA methylation the core histones in the dimers with histone variants such can be reversed by the ten-eleven translocation (TET) as histone H2A.Z or H3.3 (Clapier & Cairns 2009, Szenker family of methylcytosine hydroxylases that convert et al. 2011). In addition to controlling gene transcription, 5mC to 5-hydroxymethylcytosine (5-hmC) (Li & Zhang histone variants and their PTMs also contribute to 2014). Also, drugs such as 5-azacitidine (5AC) and 5-aza- nucleosome and chromatin structure and function 2′-deoxycytidine (DAC) can block DNA methylation by (Szenker et al. 2011). directly inhibiting DNMTs (Sato et al. 2017).

The histone modification H3K4me3 has been the potential benefit of DNA methylation inhibitors in shown to protect CpG islands from DNA methylation treating PNETs, pituitary tumors and parathyroid tumors. resulting in an antagonism between H3K4me3 and DNA methylation to regulate gene transcription (Cedar & Non-coding RNA interactions Bergman 2009). Also, DNA methylation in transcription factor recognition sequences can alter transcription factor Non-coding RNAs (ncRNAs), as the name indicates, lack binding (Barlow & Bartolomei 2014). A study conducted open reading frames (ORFs) and do not encode proteins. in MIN6 mouse insulinomas cells (Miyazaki et al. 1990) Short ncRNAs such as microRNA (miRNA) are processed showed that overexpression of menin increased the from longer primary transcripts into small single-stranded expression of the lncRNA Meg3 (Modali et al. 2015). This molecules (20–24 nucleotides). They perform RNAi- effect coincided with H3K4me3 at the Meg3 promoter, and mediated (RNA interference-mediated) gene silencing hypomethylation of the central CpG of a cAMP response by complementary base-pair interactions targeting DNA element (CRE) that binds the transcription factor CREB; in chromatin for RNA-induced transcriptional silencing the DNA hypomethylation prevented CREB binding (RITS), or messenger RNAs (mRNAs) post-transcriptionally (Zhao et al. 2006, Modali et al. 2015). Whether similar for degradation or by blocking translation (Cech & Steitz instances of reciprocal H3K4me3 and DNA methylation 2014). Long non-coding RNAs (lncRNAs) are transcripts together with altered transcription factor binding are of 200 bp or more in length that can interact with DNA present in MEN1-associated endocrine cells remains to be and proteins whereby they also participate in epigenetic determined. regulation (Kopp & Mendell 2018). Some lncRNAs have Several studies have shown indirect evidence for the been shown to reside in chromatin-modifying and participation of DNA methylation in MEN1-assocated chromatin-remodeling protein complexes that silence endocrine cell types. DNA hypermethylation has been gene expression. The eukaryotic ncRNAs play a dual role shown to occur in a subset of inherited MEN1 tumors, in epigenetic regulation – some miRNAs can silence genes sporadic PNETs, sporadic pituitary tumors or sporadic that encode epigenetic factors such as DNMTs, and some parathyroid tumors at the promoter region of different lncRNAs can directly participate in chromatin-modifying genes (APC, CTNNB1, CDKN2A/p16, CDKN2B/p15, epigenetic control mechanisms such as in the chromatin HIC1, MEG3, MLH1, RASSF1A and RIZ1), with some also inhibitory complex polycomb group (PcG) repressive correlating with respective reduction of gene expression complex 2 (PRC2) (Fabbri et al. 2007, Kaneko et al. 2014, (Carling et al. 2003, Zhao et al. 2005, Lindberg et al. Chen et al. 2015, Mondal et al. 2015). 2008, Juhlin et al. 2010, Svedlund et al. 2012, Modali In endocrine cell-line models (MIN6 and βlox5 cells), et al. 2015). A genome-wide analysis of quantitative DNA microRNA-24 (miR24) has been shown to target menin, methylation in parathyroid tissue samples showed global and increased miR24 level was also shown to correlate DNA hypermethylation in menin-null parathyroid tumors with decreased menin level in the islets of Men1f/f;RIP-Cre from MEN1 patients, and increased transcript levels of mice and human parathyroid tumors (Luzi et al. 2012, DNMT1, predicting increased DNMT1 activity in menin- Vijayaraghavan et al. 2014, Grolmusz et al. 2017, Luzi et al. null parathyroid cells accounting for the hypermethylated 2017). This silencing of menin by a microRNA predicts DNA (Yuan et al. 2016). Another study showed altered DNA downstream effects on chromatin modifications because methylation profiles in normal and pathologic parathyroid menin acts as an epigenetic co-factor in chromatin- tissue with gene expression changes, and restored modifying protein complexes. Low levels of the lncRNA expression of hypermethylated genes upon treatment of MEG3 have been observed in mouse and human MEN1- primary cell cultures of parathyroid tumors with the DNA associated PNETs, human sporadic PNETs, and in human methylation inhibitor DAC (Starker et al. 2011). A few sporadic pituitary tumors (Zhao et al. 2005, Modali studies in endocrine/endocrine-related cell lines (BON1, et al. 2015). In MIN6 mouse insulinoma cells, Meg3 was H727, MIN6) have reported growth inhibition upon demonstrated to co-immunoprecipitate with Ezh2, which treatment with DNA methylation inhibitors 5AC or DAC is part of PRC2 for H3K27me3 establishment (Iyer et al. (Alexander et al. 2010, Modali et al. 2015). These studies 2017). How loss or gain of these ncRNA interactions suggest DNA hypermethylation as an oncogenic event affect endocrine tumor epigenetics should be further in MEN1-associated endocrine cell types, and therefore, investigated.

Targeting the pathologic epigenome of Perspectives MEN1-associated endocrine cell types In this review, we aimed to survey the literature for Epigenome profiling for global epigenetic changes, such evidence of epigenetic regulation in ‘MEN1-associated as in DNA methylation or histone modifications between endocrine cell types’, which mainly included information normal and pathological samples, has demonstrated from the parathyroids, pituitary, pancreatic islets, the presence of epigenetic changes in tumors and other and lung neuroendocrine cells with the intention of disease states that could be exploited as targets for therapy highlighting the direct effects of menin as an epigenetic (Jones et al. 2016). Epigenetic therapies that are under co-factor or functionally related epigenetic effects. Whole- investigation to revert specific disease epigenomes to genome/exome sequencing efforts have identified somatic normal are designed to block the enzymatic activity of mutations in the MEN1 gene and/or genes associated with epigenetic regulators, disrupt interactions in chromatin- epigenetic control mechanisms in sporadic endocrine modifying protein complexes or target specific epigenetic tumors – non-functioning and functioning PNETs, factors for degradation (Ahuja et al. 2016). However, parathyroid adenoma and lung neuroendocrine tumors. designing of such therapeutics has to overcome several Future studies directed toward identifying the downstream issues and challenges such as cell-specific targeting due effects from these mutations will help to understand the to heterogeneity of the affected tissues, side effects, contribution of epigenetic changes in the pathogenesis of drug resistance and achieving constant, consistent and the specific endocrine tumors. Work conducted in cell lines long-lasting inhibition of the target (Dawson 2017). derived from pancreatic or lung neuroendocrine tumors Potential therapies in the form of various small molecule or their metastasis, and in vivo work in insulinomas of chemical inhibitors that target epigenetic enzymes (e.g., Men1f/f;RIP-Cre mice have established the role of menin as DNMTi, HDACi, KTMi, KDMi, and BETi) have been an epigenetic co-factor in the context of a few epigenetic developed. DNA hypomethylating agents (decitabine and modifications: H3K4me3, H3K9me3 and H4R3me2s and azacitidine) that target DNMT1 and DNMT3A, and a few DNA methylation. Given that DNA methylation and HDACi (panobinostat, vorinostat, and romidepsin) were multiple histone modifications are modulated by menin approved by the US Food and Drug Administration (FDA) raises the question whether epigenetic changes in MEN1- for the treatment of specific hematologic malignancies associated tumors would encompass multiple epigenetic (Ahuja et al. 2016). marks. Future studies directed toward determining the As discussed earlier, various HDACi, BETi and DNMTi changes in multiple histone modifications and/or DNA have been shown to inhibit the growth of endocrine/ methylation in tumors (epigenome signatures) compared endocrine-related cells lines, and intraperitoneal injection to corresponding normal cells could be valuable for of the BETi JQ1 has been shown to reduce cell proliferation understanding the tumorigenesis process and potential and increase apoptosis in insulinomas of the Men1f/f;RIP- treatment opportunities. The challenges for conducting Cre mouse model (Baradari et al. 2006, Adler et al. 2009, such studies for MEN1-associated tumors are the non- Alexander et al. 2010, Wong et al. 2014, Modali et al. availability of ‘corresponding’ specific normal cell types, 2015, Arvidsson et al. 2016, Lines et al. 2017). Another and the lack of relevant cell lines from human tumors. study showed that lack of a histone demethylase Rbp2 Perhaps generating mouse xenografts of human tumors in the Men1f/f;RIP-Cre mouse background could decrease could prove to be useful in establishing cell lines. A recent insulinoma development (Lin et al. 2011). Rbp2 loss study has shown that in the context of human PNETs could potentially restore H3K4me3 that occurred in the using a human hepatocyte growth factor (HGF) ligand insulinoma cells due to menin deficiency. Furthermore, facilitated the successful generation of xenografts in mice overexpression of RBP2 was detected in human PNETs because PNETs express high levels of the HGF receptor or their metastases, implicating a target for histone c-MET (Krampitz et al. 2016). Similar methods could demethylase inhibitor therapy (Maggi et al. 2016). These be used to establish cell lines from MEN1 and sporadic studies suggest the potential of epigenetic drug therapy tumors. Such tumor models and cell lines will facilitate of tumors arising in MEN1-associated endocrine cell a deeper understanding of epigenetic changes during the types irrespective of whether the epigenetic status of the tumorigenesis process to exploit epigenetic therapy as tumor cells is a direct consequence of menin loss or a a treatment option for the tumors of MEN1-associated consequence of the tumorigenesis process. endocrine cell types.

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Received 18 February 2018 Accepted 3 April 2018 Accepted Preprint published online 3 April 2018